The present invention relates to optical sensors which rapidly detect bacteria and other microorganisms, as well as to methods for enhancing signals in optical bacterial sensors. The sensors detect and discriminate classes of mircroorganisms using semi-selective interactions of luminescent compounds and molecular receptors with the microorganisms.
In many cases there is a need for very rapid detection of biological substances, such as bacteria, viruses, rickettsia, fungi, other microorganisms, and their fragments. This is important for medical diagnosis as well as for agriculture, food processing, bioprocessing, water purification, and detection of biological weapons to prevent harm to a civilian population. Current detection methods include cell culture, microscopy, immunoassay, nucleic acid probes, and optical detectors. Assay times vary from minutes to days. Only culture and polymerase chain reaction (PCR) based tests are very sensitive. Culture and microscopy depend on isolating the intact microorganisms from the milieu to be tested. For culture, the cells must be viable, but some microorganisms are viable but not culturable. Culturing and microscopic enumeration can take several days. Tests based on genetic methods, including PCR, require the presence of intact deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), must be done in the laboratory, and the instrumentation is costly.
In immunoassay and immunofluorescence stain assays, a complex is formed between the antibody, the analyte recognized (from or on the microorganism) and a label or signal generator (i.e., an enzyme) that can be measured. The measurement may represent the formation of a complex, such as in sandwich immunoassays, or the lack of formation of complex, as in most competitive immunoassays. Binding of the label or signal generator to the analyte is via the antibody.
In a competitive assay, the label or signal generator is bound to an antigen similar to the analyte. As the analyte competes with the labeled antigen for binding to the antibody, the amount of signal changes. In this case as well, the label never directly attaches to the analyte.
Optical waveguide fibers have been demonstrated in the laboratory for detecting many different chemical parameters. Many of the sensors are based on the use of the used immunoassays. For example, Hirschfield, in U.S. Pat. No. 4,447,546, disclosed the use of optical fibers as waveguides which capture and conduct fluorescence radiation emitted by molecules near their surface. Thompson et al., in U.S. Pat. No. 5,061,857, disclose an optical waveguide binding sensor having improved sensitivity for use with fluorescence assays. In these patents, the analyte is specifically labeled such that the antibody analyte complex is formed on the optical fiber waveguide is detected from the fluorescent signal excited and guided toward a fluorimeter using the evanescent wave portion of the optical fiber.
Walt et al., in U.S. Pat. 5,244,813, describes the use of fiber optic sensors for detecting organic analytes in samples.
Sato, in U.S. Pat. No. 5,766,868, describes a hydrophobic membrane to obtain a count of viable microbes in industrial water, raw materials, intermediates, and products processed in the food and beverage, pharmaceutical cosmetic, and microelectronic industries. In this case a hydrophobic filtration membrane is used under conditions to contain and confine the individual microbes or colony forming unit on the surface of the membrane to allow individual detection of the suspected microbes. Microbes are detected as bright spots representing their existence individually (i.e., without cultivation), or as a colony forming unit formed after cultivation of bacteria after filtration.
Optical fibers and optical fiber strands have been used in combination with light energy absorbing dyes for medical, environmental, and chemical analytical determinations. The optical fiber strands used for analytic determinations typically are glass or plastic extended rods having a small cross-sectional diameter. When light energy is projected into one end of the fiber strand (the proximal end), the angles at which the various light energy rays strike the surface and are reflected are greater than the critical angle. These propagated rays are piped through the length of the fiber strand by successive internal reflections, and eventually exit from the opposite end of the strand, the distal end. Typically, bundles of these strands are used collectively as optical fibers in a variety of different applications.
Typically, light from an appropriate energy source is used to illuminate the proximal end of an optical fiber or a fiber bundle. The light propagates along the length of the optical fiber and a portion of this propagated light energy exits the distal end of the optical fiber and is absorbed by one or more light energy absorbing dyes. The light energy absorbing dye may or may not be immobilized, may or may not be directly attached to the optical fiber itself, may or may not be suspended in a fluid sample containing one or more analytes of interest to be detected, and may or may not be retainable for subsequent use in a second optical determination.
Once the dye has absorbed the light energy, some light energy of varying wavelength and intensity typically returns through the distal end of the optical fiber and is then conveyed through either the same fiber or a collection of fibers to a detection system where the emerging light energy is observed and measured. The interactions between the incoming light energy conveyed by the optical fiber and the properties of the light absorbing dye, both in the presence of a fluid sample containing one or more analytes of interest and in the absence of any analytes whatsoever, provide an optical basis for both qualitative and quantitative spectral determinations.
Because of the photonic, optoelectric and microcircuitry and enhanced video technology now available, a variety of light image processing and analytical systems exist which can be used to enhance, analyze, and mathematically process the light energies introduced to and emerging from the absorbing dyes in these optical analytical techniques. Typically, these systems provide components for image capture, data acquisition, data processing and analysis, and visual presentation to the user. Commercially available systems include the QX-7 image processing and analysis system sold by Quantex, Inc. of Sunnyvale, Calif., and the IM Spectrofluorescence imaging system offered by SPEX Industries, Inc. of Edison, N.J., the miniature fluorometer offered by Ocean Optics, Inc. of Sunnyvale, Calif. Each of these systems can be combined with microscopes, cameras, and/or television monitors and computer interface for automatic processing of all light energy determinations.
Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light energy at specified wavelengths (excitation frequency) and them emit light energy of a longer wavelength and at a lower energy (emission frequency). This is referred to as fluorescence if the emission is relatively long-lived, typically on the order of 1011 to 107 seconds. Substances able to fluoresce share and display a number of common characteristics: they absorb light energy at one wavelength or frequency to reach a xe2x80x9csingletxe2x80x9d, an excited energy state, and subsequently emit light at another light frequency, returning to a xe2x80x9cgroundxe2x80x9d energy level. The absorption and fluorescence emission spectra are thus individual for each fluorophore, and are often graphically represented as two separate curves which are slightly overlapping.
All fluorophores demonstrate the Stokes"" shift, i.e., the emitted light is always at a longer wavelength relative to the wavelength of the excitation light and at a lower energy level relative to the wavelength and energy level of the exciting light absorbed by the substance. Moreover, the same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within the limits. The light emitted by the fluorophore will always provide the same emission spectrum as emerging light. Finally, fluorescence may be measured as the quantum yield of light emitted. The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore.
Other optical phenomena which can be used to detect microorganisms include general luminescence and chemiluminescence. Luminescence is light emitted from longer lived energy state as exemplified by luminescent lanthanides used to detect bacterial spores. [Rosen, D.L., Sharpless, C., and McGown, L.B., xe2x80x9cBacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence,xe2x80x9d Anal. Chem., 69, 1082-1085 (1997).]
Chemiluminescence, on the other hand, refers to compounds which upon subjection to a chemical reaction, such as exidation or reduction, emit light. The intensity of the emitted light is proportional to the concentration of the agent which acted upon the chemiluminescent compound. Chemiluminescence labels for immunoassays and nucleic acid probe assays provide a high degree of sensitivity when compared to other commonly used labels. For an overview of the subject see McCapra et al., Journal of Bioluminescence and Chemiluminescence 4: 51-58 (1989).
Among the conventional detection methods, such as cell culture, microscopy, immunoassay and PCR analysis, none offers all of the advantages of high sensitivity, short assay times (under approximately five minutes) and low technical complexity.
It is an object of the present invention to overcome the aforesaid deficiencies in prior art.
It is another object of the present invention to provide an optical sensor for rapid, sensitive detection of microorganisms without laboratory manipulation.
It is another object of the present invention to provide semi-selective detection and discrimination of classes of microorganisms.
It is another object of the present invention to provide methods for enhancing the signal in optical microorganism sensors.
The present invention provides a rapid, sensitive sensor for detecting microorganisms particularly air, liquid-borne, and aerosolized comprising:
Molecular receptors to interact with the microorganisms
Fluorescent or luminescent reagents and combinations thereof to report the presence of microorganisms
A polymer membrane to immobilize and stabilize the reagents
A non-drying membrane additive to provide a suitable environment for the chemical reaction
An optical substrate as a sensor support
A conformable optical substrate or rotating film to refresh the sensor chemicals
Miniature optical module including sources and light detectors, filters and lenses to detect the luminescent or fluorescence signal from the sensor.
The present invention thus provides a method for improving the detection limit and response time of the optical microorganism sensors using immobilized bioactive peptides.
The present invention also provides a method for improving the detection limit (sensitivity) of the optical microorganism sensors by Surface Enhanced Fluorescence (SEF) using immobilized metal colloid particles such as silver and gold colloidal particles in the sensing membrane.
Another method for enhancing the signal according to the present invention includes optimizing surface enhanced fluorescence using sol-gel coated colloidal particles co-immobilized in the sensing membrane.
These enhancement methods can be used singly or in any combination thereof.
The sensitivity, detection limit, and response time of optical microorganism sensors are improved in those systems which use biocidal peptides co-immobilized in the sensing membrane.
Alternatively, the signal is enhanced using surface enhanced fluorescence (SEF) using co-immobilized metal colloids such as silver and gold colloidal particles in the sensing membrane.
Another method for enhancing the signal includes optimizing surface enhanced fluorescence using sol-gel coated colloidal particles co-immobilized in the sensing membrane. These enhancement methods can be used singly or in any combination thereof.